Team:Warsaw/Calendar-Main/10 October 2008

From 2008.igem.org

(Difference between revisions)
Line 62: Line 62:
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/f/fe/Grad_arac_09_10_2008.jpg"></a>
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/f/fe/Grad_arac_09_10_2008.jpg"></a>
-
<var><b>Fig. 2.</b>Gradient PCR products for AID under pBAD/araC</var>
+
<var><b>Fig. 2.</b>Gradient PCR products for AID under pBAD/araC<br> 1< -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 40&deg;C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 60&deg;C (the highest temperature of gradient).</var>
<p class="hide"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg">
<p class="hide"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg">

Revision as of 18:50, 28 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 


Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K., Piotr

  1. Clean-up of overnight digest reaction.
  2. Digest of pSB1A3 carrying ΔA (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
  3. Gel electrophoresis and gel-out of proper band - 2200 bp. Fig. 1.
  4. Ligation of digested pSB1A3 with alpha_linker under PT7 (BBa_K103019) fragment (1 hr).
  5. Transformation of TOP10 with above ligation.
  6. Tranformants plating on LB with ampicillin.

Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K., Piotr

  1. Ligation of digested pSB2K3 vector from (30 September) with omega_linker under PT7 (BBa_K103020) fragment (1 hr).
  2. Transformation of TOP10 with above ligation.
  3. Tranformants plating on LB with kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day - pMPMT5+AID (with removed EcoRI site).
  2. Control digest of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.
  3. Temperature gradient PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 40 - 60 °C; elongation length 2.30 min) to optimize conditions for obtaining AID under pBAD/araC (BBa_K103002).
  4. Gel electrophoresis. Best annealing temperature (45 °C) chosen
  5. PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 45 °C; elongation length 2.30 min) to obtain AID under pBAD/araC (BBa_K103002)fragment.
  6. Gel electrophoresis and gel-out of proper band 1800 bp. Fig. 1.
  7. Digest of isolated PCR product with XbaI and PstI (Tango buffer).
  8. Clean-up of digested PCR product.
Fig. 2.Gradient PCR products for AID under pBAD/araC
1< -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 40°C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 60°C (the highest temperature of gradient).

Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AlphaL+SacI and AlphaP+link10+homo2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp. Fig. 1.
Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha